SHP2 inhibitors maintain TGFβ signalling through SMURF2 inhibition

Despite the promising antitumor activity of SHP2 inhibitors in RAS-dependent tumours, overall responses have been limited by their narrow therapeutic window. Like with all MAPK pathway inhibitors, this is likely the result of compensatory pathway activation mechanisms. However, the underlying mechanisms of resistance to SHP2 inhibition remain unknown. The E3 ligase SMURF2 limits TGFβ activity by ubiquitinating and targeting the TGFβ receptor for proteosome degradation. Using a functional RNAi screen targeting all known phosphatases, we identify that the tyrosine phosphatase SHP2 is a critical regulator of TGFβ activity. Specifically, SHP2 dephosphorylates two key residues on SMURF2, resulting in activation of the enzyme. Conversely, SHP2 depletion maintains SMURF2 in an inactive state, resulting in the maintenance of TGFβ activity. Furthermore, we demonstrate that depleting SHP2 has significant implications on TGFβ-mediated migration, senescence, and cell survival. These effects can be overcome through the use of TGFβ-targeted therapies. Consequently, our findings provide a rationale for combining SHP2 and TGFβ inhibitors to enhance tumour responses leading to improved patient outcomes.


H358
Rel.Absorbance at 590nM NBT-II cells transfected with siRNA targeting SHP2 or relevant controls were plated for scratch assay and treated with either DMSO, HGF (8 μM), mitomycin C (5μg/ml) or HGF (8 μM) in combination with mitomycin C (5μg/ml), panels show migration at 0, 16, or 20 hours.Representative images are shown (scale bars, 650 μm).(b).Percentage of migrated area was determined with respect to control (0 h) and a graph was plotted.*P <0.05, **P <0.01 using Student's t-test.Data are mean SD from three random fields.Data are representative of three independent experiments with similar results.(c) Immunoblot of NBT-II cells from A and B. Lysates are probed with indicated antibodies.(d) Transwell assay of H1792 cells treated with SHP099 (10 μM), HGF (8 μM), or in combination for 16 hours prior to fixation and crystal violet staining (scale bars 100 μm).(e) Graph represents percent number of migrated cells taken from four different random fields from panel (d).Data are mean ± SD of triplicate samples from a representative experiment performed three times.*P≤0.05 using Student's t-test.
cells treated with SHP099 (10 µM) or DMSO were plated for scratch assay and treated with or without HGF (8 µM), panels show migration at 0 and 26 h.Representative images are shown (scale bars, 200 µm).(b) Percentage of migrated area was determined with respect to control (0 h) and a graph was plotted.*P ≤0.05 using Student's t-test.Data are mean ± SD from three random fields.Data are representative of three independent experiments with similar results.(c) Overall cell count of H358 cells from A and B treated with or without SHP099 (10 µM) for indicated time points and relevant data was plotted.P ≤0.0001 using Student's t-test.Data are mean ± SD from nine random fields.Data are representative of two independent experiments with similar results.(d) H1792 cells treated with SHP099 (10 µM) or DMSO were plated for scratch assay and treated with or without HGF (8 µM), panels show migration at 0 and 28 h.Representative images are shown(scale bars, 200 µm).(e) Percentage of migrated area was determined with respect to control (0 h) and a graph was plotted.*P ≤0.05 using Student's t-test.Data are mean ± SD from three random fields.Data are representative of three independent experiments with similar results.(f) Overall cell count of H1792 cells from D and E treated with or without SHP099 (10 µM) for indicated time points and relevant data was plotted.P=0.03 using Student's t-test.Data are mean ± SD from nine random fields.Data are representative of two independent experiments with similar results.
c) L 65, H 58, H1792, or L 99 cells were stimulated with TGF (100 pM) or SHP099 (10 µM) as indicated for hours.PAI1 (a), p21 (b), or SMAD7 (c) mRNA levels relative to GAPDH are shown as evaluated by uantitative real time PCR.Data are shown as the mean ± SD of triplicate samples from a representative experiment performed two times.*P ≤0.05, **P ≤0.01, ***P ≤0.001, ****P ≤0.0001 as determined by Student's t-test.(d) uantification of nude mice bearing xenograft tumours of H 58 cells treated with SHP099 (75 mg/kg), Trametinib (0. mg/kg), or the combination for 50 days.Points indicate mean tumour volume, bars, S .(e) Tumours were harvested at day 50 and analy ed by immunohistochemistry for the phosphorylation of SMAD2.Representative images are shown.(f) Gene set enrichment analysis of TGF (PLASARI TGFB1 TARG TS 10HR P and R ACTOM TGF B TA SIG-NALLING ACTI AT S SMADS,) gene set signatures, extrapolated from the GS 109270 data set derived from five vehicle-and seven SHP099-treated patient derived xenograft tumours.nrichment scores ( Ss), normali ed enrichment scores (N Ss), P values, and FDR are reported.Quantification of H358 cells similar to figure 5b,c.Control (red), SHP099 (10 µM) (purple), A83-01 (10 µM) (green) or the combination A83-01 (10 µM) plus SHP099 (10 µM) (blue).Error bars in A represent SD of three independent experiments.*P ≤0.05,* of figures 5H.Data are mean ± SD from three independent experiments with similar results.(b) Quantification of figures 5H.Data are mean ± SD from three independent experiments with similar results.(c) Stacked bar graphs showing the percentage of cells in different phases of the cell cycle.Percentage of cells in subG1, G1, S, and G2 phases was calculated with Flowjo software.Results represent mean values from three independent experiments.Gene set enrichment analysis of EMT (FOUROUTAN_TGFB_EMT_UP, AND HALL-MARK_ EPITHE-LIAL_MESENCHYMAL_TRANSITION) gene set signatures, extrapolated from the GSE109270 data set derived from five vehicle-and seven SHP099-treated patient-derived xenograft tumours.Enrichment scores (ESs), normalized enrichment scores (NESs), P values, and FDR are reported.

Absorbance at 590nM
*P ≤0.01, ***P≤0.001,**** P ≤0.0001 as determined by Student's t-test.( ) Migration assay in H358 cells similar to 5E.Percentage of migrated area was determined with respect to control (0 h) and a graph was plotted.**P ≤0.01 using Student's t-test.Data are mean ± SD from three random fields.Data are representative of three independent experiments with similar results.(c) Transwell assay of H358 cells treated with SHP099 (10 µM), or in combination with or without A83-01(10 µM) for 16 hours prior to fixation and crystal violet staining (scale bars 100 µm).( ) Graph represents percent number of migrated cells taken from four different random fields from panel (c).Data are mean SD of triplicate samples from a representative experiment performed three times.*P≤0.05 using Student's t-test.(e) NBT-II cells transduced with siRNA targeting SHP2 or relevant controls were plated for scratch assay and treated with either DMSO, HGF (8 µM), or HGF (8 µM) in combination with A-83-01 (10 µM), panels show migration at 0 and 16 hours.Representative images are shown (scale bars, 200 µm).(f).Graph extrapolated from panel E represents percentage of migrated area determined with respect to control (0 h) and a graph was plotted.**P <0.01 using Student's t-test.Data are mean ± SD from three random fields.Data are representative of three independent experiments with similar results.
Table represents list of siRNAs transduced into NBT-II cells and treated with HGF.siRNA are ranked according to percent wound healing repair (%WHR).